This proposal addresses the basic functions and regulatory mechanisms involved in retroviral RNA splicing and protein processing. We will use the avian sarcoma leukosis virus (ASLV) as a model system. The long- term objective is to elucidate the molecular mechanisms involved in these processes. Human retroviruses (HTLV and HIV) represent a significant health problem and our basic research in this area may identify steps in the infectious cycle that could be targeted for therapeutic intervention. RNA Processing. Using an infectious ASLV DNA clone, we have constructed a cis-acting RNA mutation which produces an imbalance of spliced and unspliced forms of viral RNA and a concomitant replication defect. We have isolated replication competent revertant viruses that have restored balanced splicing through acquisition of second-site mutations. The location of these second-site mutations defines regions that are involved in splicing control. We plan to use these mutations to test the current models for retroviral splicing control. We will also use in vitro mutagenesis to determine if ASLV proteins are required in trans for splicing regulation. Processing of gag and pol Precursor Polypeptides by the Retroviral Protease (PR). Our proposed experiments will address: a) How the diverse cleavage sites on the precursor molecules are recognized by PR to give rise to mature virion structural proteins; b) the mechanism by which cleavage is delayed until particle formation occurs; and c) what constitutes the active site of the ASLV PR. We have adopted two well- defined systems of study: 1) PR-directed cleavage of viral precursor fragments synthesized in E. coli cells and 2) a peptide substrate assay. Using in vitro mutagenesis of the E. coli expression vector we will map the active site of the PR. We will also initiate high resolution structural studies (X-ray crystallography, NMR spectroscopy). The peptide cleavage assay will be used to determine the requirements for substrate recognition. Since protease activity is required to form infectious retrovirus particles, inhibitors of PR represent potential drugs for treatment of retroviral-induced diseases.